The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sevigny, C. P.
Right arrow Articles by Okusa, M. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sevigny, C. P.
Right arrow Articles by Okusa, M. D.
The Journal of Immunology, 2007, 178: 4240-4249.
Copyright © 2007 by The American Association of Immunologists, Inc.

Activation of Adenosine 2A Receptors Attenuates Allograft Rejection and Alloantigen Recognition1

Charles P. Sevigny2,*, Li Li2,*, Alaa S. Awad*, Liping Huang*, Marcia McDuffie{dagger}, Joel Linden*,{ddagger},§, Peter I. Lobo* and Mark D. Okusa3,*,{ddagger},§

* Department of Medicine, {dagger} Department of Microbiology, {ddagger} Cardiovascular Research Center, and § Carter Immunology Center University of Virginia, Charlottesville, VA 22908


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The current studies investigated the in vitro and in vivo effect of adenosine 2A receptor (A2AR) agonists to attenuate allogenic immune activation. We performed MLRs with spleen T lymphocytes and APCs isolated from wild-type and A2AR knockout mice of both C57BL/6 and BALB/c background strains. Two-way MLR-stimulated T cell proliferation was reduced by ATL313, a selective A2AR agonist in a dose-responsive manner (~70%; 10 nM), an effect reversed by the A2AR antagonist ZM241385 (100 nM). By one-way MLRs, we observed that ATL313’s inhibitory effect was due to effects on both T cells and APCs. ATL313 suppressed the activation markers CD25 and CD40L and the release of inflammatory cytokines IFN-{gamma}, RANTES, IL-12P70, and IL-2. ATL313 also increased negative costimulatory molecules programmed death-1 and CTLA-4 expressed on T cells. In lymphocytes activated with anti-CD3e mAb, ATL313 inhibited the phosphorylation of Zap70, an effect that was reversed by the protein kinase A inhibitor H-89. In skin transplants, allograft survival was enhanced with ATL313, an effect blocked by ZM241385. These results indicate that A2AR agonists attenuate allogenic recognition by action on both T lymphocytes and APCs in vitro and delayed acute rejection in vivo. We conclude that A2AR agonists may represent a new class of compounds for induction therapy in organ transplantation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Innate and adaptive immunity critically impacts acute and chronic allograft rejection as well as ischemia-reperfusion injury (IRI),4 and the ability to attenuate these effects could consistently enhance long-term allograft survival. T cells play a critical role in the pathogenesis of acute and chronic allograft rejection (1). Activation of T cells requires specific signaling by the interaction between the TCR and Ag presented by APCs and is also regulated by positive and negative costimulatory signals. B7 family ligands and receptors play an important role in controlling the activation and proliferation of T cells. Interaction of B7-1 (CD80) and B7-2 (CD86) expressed on APCs with CD28 expressed on T cells promotes T cell expansion and cytokine secretion (2). In contrast, interaction of B7-1 and B7-2 with CTLA-4 expressed on T cells, which is up-regulated after T cell activation, suppresses T cell activation (3, 4). Programmed death (PD)-1 expressed on T cells is another inhibitory molecule regulated by IFN-{gamma}, which binds to the APC ligands PD-L1 (B7-H1) and PD-L2 (B7-DC). PD-L1 shares 20–38% aa identity with other B7 family members (B7-1 and B7-2) expressed on APCs and nonlymphoid tissues. Upon T cell activation, up-regulation of PD-1 expression also contributes to T cell homeostasis (5). Thus, the negative costimulatory pathways CTLA-4:B7 and PD-1:PD-L down-regulate T cell-mediated alloimmunity, and studies in animals would support the concept that PD-1 and CTLA-4 can induce transplantation tolerance.

Adenosine 2A receptors (A2ARs) are a subtype of the G protein-coupled receptor family of adenosine receptors, which also includes A1R, A2BR, and A3R (6, 7), and they have tissue protective properties (8). Systemically administered A2AR agonists have been shown to reduce tissue injury associated with ischemia-reperfusion (9, 10, 11, 12). Through a series of studies, we demonstrated that the protective effect of A2AR agonists was mediated through bone marrow-derived leukocytes (13). Furthermore, additional studies indicate that A2AR agonists reduce kidney IRI through activation of A2ARs expressed on CD4+ cells (14). A2AR agonists, including ATL313, attenuate IFN-{gamma} and limit anti-CD3e mAb-induced T cell activation (15). Delayed graft function is a form of IRI that is thought to increase the immunogenicity of the transplanted allograft (8) in part due to an increase in MHC class I and II expression leading to episodes of acute rejection and chronic graft loss. These findings suggest that A2ARs may be uniquely suited to block T cell activation associated with IRI and delayed graft function. Moreover, given the diverse effects of A2AR activation on T cell function, we considered the possibility that A2AR activation attenuates allogenic recognition, thus rendering agonists of A2ARs useful in attenuating rejection in transplantation.

In the current study, we sought to determine the effect of A2AR agonists in vitro on alloantigen-induced T cell activation and proliferation as well as the effect of costimulatory molecules. We studied the effect of A2AR activation on the MLR, an assay in which the lymphocytes from two separate mouse strains are combined and cultured in vitro to initiate an alloimmune response. An advantage of this approach is that T cell activation occurs under physiological conditions that involve TCR activation and Ag presented by dendritic cells (DCs) and that are regulated by positive and negative costimulatory molecules. In vitro studies demonstrate that, in response to T cell activation by alloantigens, A2AR agonists attenuate T cell activation and decrease secretion of proinflammatory cytokines through direct and independent effects on both T cells and APCs. Importantly, the A2AR agonist increased expression of negative costimulatory molecules PD-1 and CTLA-4 that are involved in peripheral tolerance. Lastly, we demonstrated that the in vitro inhibitory effects of A2AR agonists on alloantigen-induced immune response likely contribute to the attenuation of tissue rejection following skin transplantation. These results suggest that A2AR activation is a unique and potent strategy in attenuating IRI and delaying allograft rejection following organ transplantation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

All animals were handled and procedures were performed in adherence to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and in accordance with the University of Virginia Animal Care and Use Committee protocols. C57BL/6 (B6) and BALB/c mice were purchased from Charles River Laboratories. The source and derivation of congenic A2AR knockout (KO) mice was described previously (13, 16). To generate Adora2anull (A2ARKO) mice congenic to BALB/c (17, 18, 19), a mapping panel of 55 microsatellite loci informative for a (C57BL/6J x BALB/cByJ) cross was identified with coverage for every chromosome, except X and Y, at a density of <30 cM. The mapping panel is available on request (mjm7e@virginia.edu). A single founder male was mated to BALB/cByJ female mice. (BALB/c x B6.129-Adora2atm1jfc)F1 males carrying the Adora2a mutation were then mated to BALB/cByJ females in two successive generations of backcrossing with breeders selected for maximal BALB/cByJ homozygosity. The BALB/cByJ Y chromosome was introduced by crossing BALB/cByJ males to genotypically selected F1N3 females carrying the Adora2a mutation in the final backcross generation before initiation of inbreeding with F1N4 mice of both sexes. Residual B6 or 129 alleles in the congenic line were detected only on mouse chromosome 10 between Adora2a and D10Mit35 (74.77–121.60 Mb, respectively; NCBIm36).

Harvesting of mouse splenocytes

Spleens were extracted from B6 and BALB/c mice and disrupted under sterile conditions in PBS through 40-µm BD Falcon cell strainers (Fisher Scientific). Leukocytes were then isolated via density gradient centrifugation using Histopaque 1083 (Sigma-Aldrich) and washed in RPMI 1640 supplemented with 10% heat-inactivated FBS (Invitrogen Life Technologies) and 1% antibiotic/antimitotic solution (Invitrogen Life Technologies). Cells were resuspended (1 x 106 cells/ml) in the culture medium in the presence of adenosine deaminase (1 U/ml; Roche Diagnostics).

Activation of lymphocytes by alloantigens by MLRs

In two-way MLR assays, 2 x 105 leukocytes from B6 mice in 0.1 ml were cocultured with an equal number of leukocytes from BALB/c mice using 96-well flat-bottom tissue culture-treated plates. Appropriate compounds were administered at the initiation of the two-way MLR assay. All compounds were diluted in 10 µl of medium before being added to the appropriate wells, and cells were allowed to incubate at 37°C in the presence of 5% CO2. As controls, we added vehicle without compound to cell cultures. Second, in two-way MLR assays, cells obtained from each murine strain were cultured alone, i.e., without mixing, so as to calculate the added effect obtained with cocultures.

For one-way MLR studies, cell cultures were prepared as described previously (20). Stimulator cells were prepared from spleens of wild-type (WT) (B6 background) or A2AKO (B6 background) mice. Spleens were cut into small pieces and digested with 1 mg/ml collagenase type IA (Sigma-Aldrich) for 10 min at 37°C, which can increase the yield of APCs. Single-cell suspension was treated with mitomycin C (50 µg/ml; Sigma-Aldrich) for 20 min at room temperature and then washed twice with RPMI 1640. Responder spleen T cells from spleens of WT (BALB/c background) or A2AKO (BALB/c background) mice were harvested by negative isolation using magnetic beads, according to the manufacturer’s protocol (Dynal T Cell Negative Isolation kit; Invitrogen Life Technologies). The purity of the CD3 T cells was measured by FACS (BD Biosciences), and T cells with >98% purity were used in this study. Responder (2 x 105) and stimulator (4 x 105) cells were added to round-bottom 96-well plates to a final volume of 200 µl of RPMI 1640 with 10% FCS/1% antibiotic/antiamitotic solution and in the presence of adenosine deaminase (1 U/ml; Roche Diagnostics). Each experiment was performed in triplicate.

Vehicle or 4-[3-[6-amino-9-(5-cyclopropylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl]-piperidine-1-carboxylic acid methyl ester (ATL313) was used in two-way (0.01–100 nM) and one-way (10 nM) MLRs. 4-(2-[7-Amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol (ZM241385) (100 nM) was used to determine specificity of ATL313 for the A2AR.

Activation of lymphocytes by anti-CD3e

In these studies, leukocytes were activated with anti-CD3e mAb. Functional grade hamster anti-mouse CD3e mAb (clone 145-2C11; eBioscience) was used in an insoluble form (by precoating tissue culture-treated plates) or in soluble form. Plates were coated by covering wells with Ab in PBS (2 µg/ml) for 4 h at 37°C, then rinsed twice with PBS before adding cells. Soluble Ab was used at different concentrations at the initiation of the culture. Controls were the same as the two-way MLR assay.

Proliferation assays

Lymphocytes activated in an MLR or by anti-CD3e mAb were allowed to incubate up to 3 days before harvesting. [3H]Thymidine (0.5 µCi/well; MP Biomedicals) was added 18–24 h before harvesting (Skatron Instruments) using Type A filter mats (PerkinElmer Life and Analytical Sciences) and beta plate scintillation mixture (PerkinElmer). Disintegrations per minute were determined using a liquid scintillation counter (1205 Betaplate; PerkinElmer).

Multiplex bead array for quantitating cytokines

Multiplex bead array was performed on supernatants obtained on day 3 of culture. Microbead labeling was performed using the Bio-Plex multiplex cytokine assay kit (Bio-Rad) in accordance with manufacturer’s protocol, and cytokine levels were analyzed on the Bio-Plex system (Bio-Rad).

ELISA for quantitating IFN-{gamma}

ELISA was performed on supernatants obtained on day 3 of culture. The ELISA for IFN-{gamma} (IFN-{gamma} Ready, Set, Go! ELISA kit; eBioscience) was performed according to manufacturer’s protocol and analyzed on a Model 680 microplate reader (Bio-Rad).

Multiprobe RNase protection assay

Total RNA was prepared using RNAzol B (Leedo Medical Laboratories) and analyzed by 1.5% agarose gel electrophoresis to assess the integrity of RNA before solution hybridization. Cytokine mRNA expression was assessed by BD RiboQuant Multiprobe RNase protection system (BD Pharmingen), according to the manufacturer’s protocol. In brief, mRNA-specific RNA probes were labeled with 32[P]UTP using multiprobe template sets (mCK3b; BD Pharmingen) for cytokine genes. Total cellular RNA was subjected to solution hybridization with each probe set. Hybridization was performed at 56°C before RNase treatment. Following RNase treatment, protected fragments were separated by gel electrophoresis on 5% polyacrylamide gels and exposed to Kodak X-Omat AR film at –70°C with a single intensifying screen. Band densities were quantitated using the Personal Densitometry SI (GE Healthcare), and data were analyzed by ImageQuant 5.2 (GE Healthcare).

Flow cytometry

All flow cytometry was performed on a BD FACSCalibur flow cytometer (BD Biosciences), and data were analyzed with FlowJo software 4.2 (Tree Star). Most of the Abs were purchased from eBioscience.

Apoptosis/Necrosis

An Annexin VFITC apoptosis detection kit (BD Pharmingen) was used to analyze MLR-activated lymphocytes after 72 h of culture with the control vehicle or ATL313 (10 nM) with or without ZM241385 (100 nM). Cultured cells (1 x 106 cells/ml) were washed with cold PBS and resuspended in annexin V binding buffer. The cell suspension (100 µl) was mixed with annexin V (5 µl) and propidium iodide (5 µl) in a 5-ml culture tube and incubated for 15 min in the dark at room temperature. A total of 400 µl of binding buffer was added to each sample before analysis by flow cytometry.

CD4/CD25 and CD4/CD40L

Three-day two-way MLR-activated cells were washed with 1% BSA/PBS and labeled with anti-mouse CD4-allophycocyanin (GK1.5; 4 µg/ml) and either anti-mouse CD25-PE (PC61.5; 4 µg/ml) or anti-mouse CD40L-PE (MR1; 4 µg/ml) in 25 µl of 0.2% BSA/PBS after blocking FcRs by CD16/32 (2.4G2; eBioscience) for 30 min on ice. Isotype controls were also performed. Samples were washed with 0.2% BSA/PBS and 2 µg/ml 7-aminoactinomycin D (Invitrogen Life Technologies) before analysis by flow cytometry.

CD4/P-Zap70

Spleen cells were isolated from B6 mice as described above, then incubated with soluble anti-CD3e mAb (145-2C11; 10 µg/ml) to stimulate lymphocytes (1 x 106) for 5 min with or without ATL313 (10 nM) or with or without ZM241385 (100 nM). Due to the short reaction time in the presence of soluble anti-CD3e mAb, the cells were pretreated with the compounds for 30 min before stimulation with the Ab. Adding 4 volumes of cold PBS terminated the reaction. Cells were collected by centrifugation and then fixed with 2% formaldehyde for 10 min at room temperature, centrifuged again, and rinsed once with PBS. The supernatant was discarded, and the tubes were chilled on ice for 1 min. Ice-cold methanol (90%) was added slowly during gentle vortexing before incubating the suspension for 30 min on ice. Cells were centrifuged and resuspended in 0.5% BSA/PBS and anti-mouse CD16/32 (2.4G2; 10 µg/ml) for 10 min at room temperature to block nonspecific binding. Rabbit anti-human P-Zap70 (1/100; Cell Signaling Technology) was added and incubated for 30 min at room temperature. Cells were rinsed as before in blocking buffer, centrifuged, and resuspended in goat anti-rabbit IgG-PE (1/2000; Southern Biotechnology Associates) in blocking buffer for 30 min at room temperature, rinsed, resuspended in 200 µl of cold PBS, and analyzed by flow cytometry. Isotype and secondary Ab controls were also performed at the same time.

PD-1 and CTLA-4

Cells activated for 3 days with soluble anti-CD3e mAb (145-2C11; 10 µg/ml) were washed with 0.2% BSA/PBS, incubated with rat anti-mouse CD16/32 (2.4G2; 10 µg/ml) to block FcRs, and added hamster anti-mouse CD3e-Alexa 647 (500A2, 4 µg/ml; Invitrogen Life Technologies) and PD-1-PE (RMP1–30; 4 µg/ml), CD4-allophycocyanin (GK1.5), or PD-1-PE (RMP1–30; 4 µg/ml) for 30 min on ice. Appropriate isotype control (hamster IgG-Alexa 647, rat IgG2b-PE, rat IgG2a; 4 µg/ml each) was used. For intracellular T cell CTLA-4 staining, cell surface was stained with hamster anti-CD3-Alexa 647 (500A2, 4 µg/ml; Invitrogen Life Technologies), washed, fixed, and permeabilized for 30 min using the BD Cytofix/Perm kit (BD Biosciences). Cells were then washed using 1x permeabilization buffer, and hamster anti-mouse CTLA-4-PE (UC10-4B9; 4 µg/ml) was added on ice for 30 min. Isotype controls using hamster IgG-Alexa 647 and hamster IgG-PE were also performed accordingly. Cells were washed and suspended in 0.2% BSA/PBS before running the sample on FACSCalibur.

Skin transplantation

Experiments were conducted in B6 mice (recipient) and BALB/c mice (donor) (7–8 wk of age; Charles River Laboratories) and were allowed free access to food and water until the day of surgery. Mice were anesthetized with a regimen that consisted of ketamine (100 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) and were placed on a thermoregulated pad to maintain body temperature at 37°C. Full-thickness tail skin (1 cm2) from donor mice was grafted on the dorsal flank area of recipient mice. The skin grafts were secured with paraffin-embedded gauze and with adhesive tape for 5 days. Graft survival was assessed by visual inspection from day 6 to day 9 in a masked fashion. The measured parameters were 1) necrosis, 2) loss of viable tissue, and 3) hair growth. Rejection was diagnosed when the graft loss was ≥70%. At the end of the experimental period, animals were euthanized, and skin graft was removed for H&E staining. Sections were viewed using a Zeiss AxioSkop microscope, and digital images were taken using a SPOT RT Camera (software version 3.3; Diagnostic Instruments). A semiquantitative score was assigned based on the masked reading for thickness of the epidermis (0–2) in which 2 = normal; 1 = loss of <50% thickness; and 0 = loss of >50% thickness and the degree of inflammation (0–5) in which score 0 = normal; 1 = <20%; 2 = 20–40%; 3 = 40–60%; 4 = >60–80%; and 5 = >80% inflammation. Skin allograft recipients were treated with vehicle or ATL313 (1 ng · kg–1·min–1) alone or combined with ZM241385 (5 ng · kg–1·min–1) via osmotic pump (ALZA) (n = 8 for each group).

Statistical analysis

Statistical analysis of all dose-response curves was analyzed by two-way ANOVA. Tukey or Bonferroni post hoc analysis was performed to assess significance at specific concentrations in these curves as well as for mean channel fluorescence (MCF) comparisons. Least significant difference post hoc analysis and paired and unpaired Student’s t tests were also used in some analyses. All curves were fit by nonlinear regression, sigmoidal dose-response parameters. Kaplan-Meier survival curve was used to assess the difference between allograft survival. All statistical analyses were performed using GraphPad Prism version 4.0 (GraphPad). A value of p < 0.05 was used to determine significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
ATL313 attenuates T cell proliferation

In the two-way MLR assay, maximum proliferation of cells was observed by day 3 of culture. Hence, we used this time point to evaluate the effect of various compounds on two-way MLR-induced cell proliferation.

Two-way MLRs were performed in the absence or presence of ATL313 (0.01–100 nM). ATL313 reduced proliferation in a dose-dependent manner with maximum inhibition achieved at a dose of 10 nM (33% of vehicle control, p < 0.001, n = 8). The addition of the A2AR antagonist ZM241385 (100 nm) attenuated the effect of ATL313 (10 nm; 83% of vehicle control, p < 0.001, n = 6; Fig. 1a).


Figure 1
View larger version (7K):
[in this window]
[in a new window]

  		FIGURE 1. ATL313 activates A2ARs and reduces lymphocyte proliferation. a, The effect of ATL313 on the proliferative response in two-way MLRs was examined in the absence (solid line, n = 8) and presence (dashed line, n = 6) of ZM241385 (100 nm). Values are means ± SEM and are calculated as percent increase in proliferation of cells compared with control vehicle-treated cells. Under vehicle conditions, [3H]thymidine incorporation in two-way MLRs, a measure of cell proliferation, ranged from 10,000 to 20,000 dpm/well. b, Reduction of proliferation by ATL313 in anti-CD3e mAb-activated lymphocytes. The effects of ATL313 on lymphocyte proliferation were examined after 24 h of activation by plate-bound anti-CD3e mAb in both WT (solid line, n = 4) and A2ARKO (dotted line, n = 4) B6 background splenocytes. Values depict mean normalized percentages between control anti-CD3e mAb-induced proliferation (vehicle-treated) and nonactivated lymphocytes (0%) ± SEM. Activated lymphocytes under vehicle treatment demonstrated a range of ~20,000–30,000 dpm/well.

 
The effect of ATL313 was not the result of an increase in apoptosis/necrosis as revealed by annexin V and propidium iodide labeling, markers for apoptosis and necrosis, respectively. There was no significant difference in the level of apoptosis or necrosis when cells were cultured without alloantigen activation (control) or when cells were activated in a two-way MLR. Similarly, the addition of ATL313 and/or ZM241385 did not increase apoptosis/necrosis (data not shown).

To confirm that the attenuation by ATL313 on lymphocyte proliferation was due to activation of A2ARs, we performed anti-CD3e mAb-induced T cell proliferation by using leukocytes obtained from WT B6 and A2AKO mice (B6 background). In splenocytes obtained from WT B6 mice and stimulated for 24 h with anti-CD3e mAb, ATL313 (0.01–100 nM) reduced lymphocyte proliferation. The effect was maximal at 10 nM (51% of control, p < 0.001, n = 4). In splenocytes obtained from A2ARKO mice, ATL313 (0.01–100 nM) had no effect (Fig. 1b). These results indicate that lymphocyte proliferative responses induced by alloantigens or anti-CD3e mAb are markedly attenuated by specific activation of A2ARs.

ATL313 inhibits lymphocyte proliferation by action on both T lymphocytes and APCs in one-way MLRs

The ATL313-specific effect on T cell and APC A2ARs was examined by using A2AR KO mice. In this one-way MLR system, T cells were harvested from BALB/c background WT and/or A2AR KO mice; APCs were harvested from B6 background WT and/or A2aR KO mice. The purity of the negative isolation T cells was >98%. We performed four groups of one-way MLRs: responder WT x stimulator WT (T cell BALB/c x APC B6), responder KO x stimulator KO (T cell BALB/c KO x APC B6 KO), responder KO x stimulator WT (T cell BALB/c KO x APC B6), and responder WT x stimulator KO (T cell BALB/c x APC B6 KO). In one-way MLRs in which both responder and stimulator cells expressed A2ARs (T cell BALB/c x APC B6), ATL 313 reduced T cell proliferation by 95% (p < 0.01; n = 4; Fig. 2a). This result is consistent with the two-way MLR result observed in Fig. 1a. When both responder and stimulator cells lacked A2ARs (T cell BALB/c KO x APC B6 KO), ATL313 had no significant effect on T cell proliferation (p = NS; n = 4; Fig. 2b). When responder cells (T cell BALB/c KO x APC B6) or stimulator cells (T cell BALB/c x APC B6 KO) lacked A2ARs, ATL313 reduced T cell proliferation by 38% (p < 0.05, n = 4; Fig. 2c) or 53% (p < 0.001, n = 4; Fig. 2d), respectively. The effect of ATL313 on T cell proliferation was blocked by the A2AR antagonist ZM241385 (Fig. 2, a, c, and d). It is interesting to note that the full effect of ATL313, apparent when both responder and stimulator cells expressed A2AR (Fig. 2a), represented an additive effect of ATL313 expressed individually on stimulator (Fig. 2c) and responder (Fig. 2d) cells. These results indicate that A2ARs expressed on both T cells and APCs mediate the inhibitory effect of lymphocyte proliferation following alloantigen immune activation.


Figure 2
View larger version (30K):
[in this window]
[in a new window]

  		FIGURE 2. ATL313 inhibits lymphocyte proliferation by action on both T lymphocytes and APCs in one-way MLRs. One-way MLRs were performed as described in Materials and Methods. Stimulator cells were prepared from spleens of WT (B6 background) or A2ARKO (B6 background) mice and responder spleen T cells were from WT (BALB/c background) or A2ARKO (BALB/c background) mice. We performed four groups of one-way MLR: responder WT x stimulator WT (T cell BALB/c x APC B6) (a), responder KO x stimulator KO (T cell BALB/cKO x APC B6 KO) (b), responder KO x stimulator WT (T cell BALB/c KO x APC B6) (c), and responder WT x stimulator KO (T cell BALB/c x APC B6 KO) (d). One-way MLRs were performed in the presence of vehicle, ATL313 (10 nM), ATL313 + ZM241385 (100 nM), and ZM 241385 alone. Experiments were performed in triplicate, n = 4 for each group. Values are mean ± SE and are normalized to vehicle group.

 
ATL313 inhibits IFN-{gamma} and cytokine release

The effects of ATL313 on two-way MLR-induced cytokine release are summarized in Table I. Using ELISA, the supernatants of two-way MLRs showed a 17-fold increase of IFN-{gamma} over unstimulated lymphocytes that were not mixed in an MLR (p < 0.001, n = 3). The increase of IFN-{gamma} release was significantly reduced in a dose-dependent manner by ATL313, an effect that was inhibited by the addition of ZM241385 (p < 0.0001, n = 3). Maximum inhibition of IFN-{gamma} release was observed with 10 nM ATL313 (21% of vehicle, p < 0.001, n = 3). ZM241385 reduced the response observed with 10 nM ATL-313 to 79% of vehicle (Table I). mRNA from the cultured lymphocytes was harvested at day 3 of culture and analyzed for steady-state IFN-{gamma} mRNA expression by RNase protection assay. Two-way MLRs produced an increase in IFN-{gamma} mRNA expression, an effect that was reduced with 10 nM ATL313. The effect of ATL313 on IFN-{gamma} mRNA expression was blocked by ZM241385 (Fig. 3). Quantitative analysis of band densities, corrected for loading (GAPDH), demonstrated that IFN-{gamma} mRNA increased in MLRs to 10.8-, 5.5-, and 10.5-fold over unstimulated cultured lymphocytes following incubation with vehicle, ATL313, and ATL313 plus ZM241385, respectively (n = 2).


View this table:
[in this window]
[in a new window]

  		Table I. Effects of A2AR agonists on two-way MLR-induced cytokine releasea

 

Figure 3
View larger version (37K):
[in this window]
[in a new window]

  		FIGURE 3. Effect of ATL313 on IFN-{gamma} mRNA expression. Total RNA was isolated from cells of two-way MLRs and subjected to RNase protection assay analysis of IFN-{gamma}. A representative photograph of this result is shown. Each lane represents solution hybridization with a radiolabeled probe for IFN-{gamma} and RNA derived from two-way MLRs. GAPDH is shown as a control for loading. Veh, vehicle; unstim, unstimulated; ATL, ATL313; ZM ZM241385.

 
Supernatant levels of IL-2, IL-12(p70), IL-12(p40), and RANTES were measured simultaneously by multiplex bead array (Table I). In these studies, cytokines in supernatants obtained from day 3 of two-way MLR cultures were compared with baseline cytokine release from unstimulated cultured lymphocytes. IL-2 levels in two-way MLRs (69.5 ± 10.0 pg/ml) were 2200% greater than those obtained from supernatants of unstimulated lymphocytes (p < 0.001, n = 3). ATL313 reduced this alloantigen activated response in a dose-dependent manner with the maximum reduction observed at a concentration of 10 nM ATL313 (60% of vehicle; p < 0.001, n = 3). This inhibitory effect was completely abrogated by the addition of ZM241385 (98% of vehicle; p = NS, n = 3).

IL-12(p70) increased 215% in MLRs (5.7 ± 0.1 pg/ml) when compared with unstimulated lymphocytes (p < 0.001, n = 3). ATL313 (10 nM) produced a dose-dependent reduction to 31% of vehicle (p < 0.001, n = 3). The addition of ZM241385 negated the inhibitory effect of ATL313 (67% of vehicle, p = NS, n = 3). ATL313 had no inhibitory effect on IL-12(p40) that had increased 214% in the two-way MLR (36.2 ± 1.2 pg/ml). Similarly, the chemokine RANTES, increased by 216% in the two-way MLR (1376.9 ± 82.8 pg/ml) when compared with unstimulated lymphocytes (p < 0.001, n = 3). ATL-313 (10 nM), reduced this effect to 67% of vehicle (p < 0.001, n = 3). ZM241385 significantly reversed this effect (90% of vehicle, p = NS, n = 3). ATL313 inhibited not only cytokines produced primarily from activated T cells (IFN-{gamma}, IL-2, RANTES) but also from activated DCs (IL-12(p70)).

We also analyzed the effect of ATL313 on a Th2 cytokine profile consisting of IL-6, IL-4, and IL-10. Over the course of 3 days, ELISA showed IL-6 steadily increased in two-way MLRs, but ATL313 did not affect the production of IL-6. IL-4 and IL-10 never reached levels detectable by ELISA over 3 days (data not shown).

ATL313 inhibits expression of CD25 and CD40L on CD4+ T cells activated in two-way MLRs

We next sought to determine the effect of A2AR agonists on the CD4+ T cell activation markers CD25 (the {alpha}-chain of the IL-2R; Fig. 4) and CD40L (a costimulatory molecule expressed on active CD4+ cells; Fig. 5). The increase in cell surface expression of CD25 on day 3 of two-way MLR (330 ± 19%, n = 4) compared with unstimulated lymphocytes was attenuated with ATL313 (10 nM; 146 ± 12%, p < 0.001 compared with vehicle, n = 4). ZM241385 (100 nM) reversed this effect of ATL313 on surface expression of CD25 (317 ± 26% of baseline, p < 0.001 compared with ATL313, NS compared with vehicle, n = 4; Fig. 4).


Figure 4
View larger version (35K):
[in this window]
[in a new window]

  		FIGURE 4. Effect of ATL313 on cell surface expression of CD25 on CD4+ T cells in two-way MLRs. a–c, Cell surface expression of CD25 (y-axis) was measured by gating on CD4+-expressing T cells (x-axis) after treatment with vehicle (a) or 10 nM ATL313 in the absence (b) and presence of 100 nM ZM241385. Levels of CD25 and CD4, as analyzed by flow cytometry, are expressed as MCF of PE and APC, respectively. d, Quantitative assessment of CD25 expression. Results represent mean of percentage of control values for unstimulated lymphocytes (100%) ± SEM; n = 4; *, p < 0.001 compared with vehicle or ATL313 + ZM241385.

 

Figure 5
View larger version (39K):
[in this window]
[in a new window]

  		FIGURE 5. Effect of ATL313 on cell surface expression of CD40L on CD4+ T cells in two-way MLRs. a–c, Cell surface expression of CD40L (y-axis) was measured by gating on CD4–-expressing T cells (x-axis) following treatment with vehicle (a) or 10 nM ATL313 in the absence (b) and presence of 100 nM ZM241385. Levels of CD40L or CD4, as analyzed by flow cytometry, are expressed as MCF of PE and APC, respectively. d, Quantitative assessment of CD40L expression. Results represent mean of percentage of control values for unstimulated lymphocytes (100%) ± SEM; n = 3.

 
Similarly, CD40L expression on the cell surface of CD4+ T lymphocytes increased to 161 ± 7% compared with unstimulated cells on day 3 of two-way MLR (p < 0.01, n = 3), and this increase in CD40L was reduced with ATL313 (10 nM; 74 ± 12% of control, p < 0.01 compared with vehicle, n = 3). The inhibitory effect of ATL313 was reversed with ZM241385 (186 ± 17% of baseline, p < 0.01 compared with ATL313, p = NS compared with vehicle, n = 3) (Fig. 5).

ATL313 inhibits anti-CD3e mAb-induced phosphorylation of Zap70

Zap70 is an intracellular tyrosine kinase that is involved in the proximal TCR signaling pathway for the T cell activation. Activation of Zap70 is coupled to downstream signaling cascades, which regulate some critical immune regulatory transcriptional gene. We reasoned that blocking the initial TCR signaling pathway with an A2AR agonist may depress the T cell immune response. Thus, we sought to determine the effect of A2AR activation on Zap70 phosphorylation. After 5 min of T cell activation by soluble anti-CD3e mAb, MCF levels of phosphorylated Zap70 increased by 186 ± 8% when compared with nonactivated cells (p < 0.001, n = 3), an effect that was reduced to 94 ± 10% of nonactivated cells by ATL313 (10 nM, p < 0.001, compared with vehicle n = 3). This inhibition of phosphorylated Zap70 by ATL313 was reversed by ZM241385 (100 nM) to 190 ± 10% of nonactivated cells (p < 0.001 from ATL313, p = NS from vehicle, n = 3). Also, the addition of H-89 (100 nm), a protein kinase A (PKA) inhibitor, blocked the effect of ATL313 by restoring the levels to 155 ± 8% of nonactivated cells (p < 0.001 from ATL313, p = NS from vehicle, n = 3). One representative example of three experiments can be seen in Fig. 6, a and b, and a summary of the MCF is illustrated in Fig. 6c.


Figure 6
View larger version (32K):
[in this window]
[in a new window]

  		FIGURE 6. Flow cytometric analysis of Zap70 phosphorylation in CD3e mAb-activated lymphocytes. a, Histogram overlays depict a rightward shift in levels of phosphorylated Zap70 (as measured by MCF of PE-labeled after activation with CD3e mAb (anti-CD3) under vehicle (veh) treatment compared with nonactivated cells, which is reversed through the addition of 10 nm ATL313 (ATL). b, In the same experiment (overlays separated for clarity), the addition of either ZM241385 (ZM; 100 nm) or the protein kinase A inhibitor, H89 (100 nm), to the ATL313-treated group causes a rightward shift of phosphorylated Zap70 compared with ATL313 alone. c, Quantitative assessment of the effect of ATL313 on phosphorylated Zap70 is shown. The graph depicts the mean of MCF, expressed as percentage of levels in nonactivated cells (100%), of phosphorylated Zap70 after 5 min of activation with soluble anti-CD3e mAb ± SEM, n = 3.

 
ATL313 induces PD-1 and CTLA-4 expression

We next sought to determine whether ATL313 could up-regulate PD-1 (Fig. 7, a–d) and CTLA-4 (Fig. 8) expression, costimulatory molecules that are important in inhibiting T cell activation. As shown in Fig. 7, a–d, on day 3 of two-way MLR, the CD3 cell surface expression of PD-1 increased compared with control unstimulated cultured cells (136 ± 10% of control, n = 9, p < 0.001). The addition of ATL313 further increased T cell PD-1 expression (192 ± 14% of control; n = 9; p < 0.001 compared with control or vehicle), an effect that was reversed by ZM241385 (134 ± 13% of control, n = 8; p < 0.05 compared with ATL313; p = NS compared with vehicle). ATL313 also had a similar effect on PD-1 expressed on CD4+ (Fig. 7, e–h) and CD8+ T cells (data not shown). Intracellular CTLA-4 expression on activated CD3 cells increased on day 3 of two-way MLR (587 ± 40% of control cells; n = 7; p < 0.001; Fig. 8). ATL313 induced expression of CLTA-4 in alloantigen-stimulated T cells (820% of unstimulated control cells, n = 7, p < 0.001 compared with control or vehicle). ZM241385 partially reversed this enhancing effect (681% of control, n = 7; p < 0.001 compared with control, p < 0.05 compared with ATL313).


Figure 7
View larger version (42K):
[in this window]
[in a new window]

  		FIGURE 7. Flow cytometric analysis of PD-1 expression on CD3+ and CD4+ cells in two-way MLRs. PD-1 levels in CD3+ (a–d)- and CD4+ (e–h)-gated events are expressed as MCF of PE. The effect of vehicle (a and e), ATL313 (10 nM; b and f), and ATL313 in the presence of ZM241385 (100 nM; c and g) on the cell surface expression of PD-1 (y-axis) and CD3+ (MCF of Alexa 647; n = 9) or CD4+ (MCF of APC; n = 3) (x-axis) is shown. Quantitative assessment of PD-1 expression is shown (d and h). Results represent mean of control values for unstimulated lymphocytes (100%) ± SEM.

 

Figure 8
View larger version (56K):
[in this window]
[in a new window]

  		FIGURE 8. Flow cytometric analysis of CTLA-4 expression on CD3+ cells in two-way MLRs. CTLA-4 levels in CD3+-gated events are expressed as MCF of PE. The effect of vehicle (a), ATL313 (10 nM; b), and ATL313 in the presence of ZM241385 (100 nM; c) on the cell surface expression of CLTA-4 (y-axis) and CD3+ (MCF of Alexa 647; n = 8 (x-axis)) is shown. Quantitative assessment (MCF of PE) of CTLA-4 expression is shown (d). Results represent mean of control values for unstimulated lymphocytes (100%) ± SEM.

 
ATL313 attenuates skin allograft rejection

Our in vitro data provide support that ATL313 inhibits T cell activation, results previously reported (15), and increases negative costimulation. We next tested the hypothesis that ATL313 can block allograft rejection in vivo in a skin transplant mouse model (Fig. 9, Table II). Skin transplants were performed in which full thickness skin grafts from BALB/c donor mice were grafted on the dorsal flank of C57BL/6 mice and treated with vehicle, ATL313 (1 ng · kg–1·min–1) alone, or ATL313 combined with ZM241385 (5 ng · kg–1·min–1) via osmotic pump (n = 8 for each group). Graft survival was assessed by visual inspection from day 6 to day 9 in a masked fashion. As shown by a Kaplan-Meier survival curve (Fig. 9, left panel), 9 days after transplantation, the graft survival rate was 12.5, 62.5, and 12.5% for vehicle, ATL313, and ATL313 plus ZM241385, respectively (n = 8 for each group; p < 0.05). At the end of the experimental period (day 9), skin grafts were harvested and prepared for histology. Photographs of representative skin transplants are shown for vehicle, ATL313, and ATL313 plus ZM241385 (Fig. 9, a, c, and e). There was loss of epidermal skin thickness as well as inflammation in the vehicle group; effects that were reduced with ATL313 treatment. The effects of ATL313 were reversed with ZM241385. Fig. 9, b, d, and f, shows representative H&E staining of the donor skin in vehicle, ATL313, and ATL313 plus ZM241385, and Table II shows semiquantitative scoring of skin thickness and inflammation.


Figure 9
View larger version (70K):
[in this window]
[in a new window]

  		FIGURE 9. ATL313 attenuates skin allograft rejection. Kaplan-Meier curve (left panel) for vehicle (black line), ATL313 (red line; ATL; 1 ng · kg–1·min–1) and ATL313 + ZM243185 (blue line; ZM; 5 ng · kg–1·min–1). n = 8 for each group; p < 0.05. Photographs (right panels) are shown for recipient mice receiving skin allografts. Photographs of transplanted skin (a, c, and e) and H & E sections (b, d, and f) are shown for vehicle (a and b), ATL313 (c and d), and ATL313 + ZM241385 (e and f). Arrowhead, donor epithelium.

 

View this table:
[in this window]
[in a new window]

  		Table II. Skin transplant histological score with H&E staina

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The current studies investigated the potential for A2AR agonists to attenuate alloantigen recognition and transplant rejection. We performed MLRs in which splenocytes were isolated from B6 and BALB/c mice and combined in vitro for 3 days with or without ATL313, a selective A2AR agonist. MLR-stimulated T cell proliferation was reduced by ATL313 in a dose-dependant manner with a maximum inhibitory response (~95%) at a dose of 10 nM, an effect reversed by 100 nM of the A2AR antagonist ZM241385. ATL313 attenuated the release from activated T cells and DCs of IFN-{gamma}, RANTES, IL-12(p70), and IL-2 but had no effect on IL-12(p40). ATL313 also inhibited the cell surface expression of T cell activation marker CD25 and CD40L induced by two-way MLRs. In contrast, ATL313 increased surface expression of negative costimulatory molecules PD-1 and CTLA-4 in two-way MLRs. In anti-CD3e mAb-activated lymphocytes, ATL313 still blocked proliferation and inhibited the phosphorylation of Zap70 induced after TCR stimulation, an effect that was reversed by the PKA inhibitor H-89. Lastly, ATL313 attenuated allograft rejection in skin transplants from donor to recipient mice. These results demonstrate that A2A agonists attenuated allogenic recognition by action on T lymphocytes and APCs in vitro, and these effects likely contribute to the attenuation of allograft rejection in vivo. Given our previous observation that A2AR agonists protected kidneys from IRI (12, 14), we believe that A2AR agonists represent a novel class of compounds ideally suited for induction therapy in organ transplantation.

The effect of A2AR agonists to attenuate T cell proliferation in two-way MLRs may be due to effects on CD4+ cells or APCs. To determine the contribution of A2ARs expressed on T cells or APCs, we performed one-way MLRs. In one-way MLRs, when A2ARs were expressed on both responder and stimulator cells, near maximal inhibition of lymphocyte proliferation was achieved. However, when A2ARs were expressed only on responder or on stimulator cells, only partial inhibition of lymphocyte proliferation was achieved. These results indicate that A2ARs expressed on both T cells as well as APCs are necessary to maximally inhibit alloantigen recognition.

Clonal expansion of T cells is mediated by the Th1 cytokine IL-2. The production of this cytokine, in conjunction with the {alpha}-chain of the IL-2R, CD25, determines whether a naive T cell will become an armed effector T cell and proliferate (21). Lymphocyte proliferation peaked in two-way MLRs at 3 days, and at this same time point, IL-2 levels increased 14-fold in two-way MLR assays over control unstimulated cultured cells. ATL313, when administered to the two-way MLR assays, reduced IL-2 production, CD25 expression, and, subsequently, proliferation. The A2AR antagonist significantly reversed the effects of ATL313 in all instances. These data imply that ATL313 is a potent anti-T cell proliferative agent. ATL313 also inhibits IFN-{gamma} expression, a cytokine that activates macrophages (22), and increases cell surface expression of MHC molecules and Ag-processing components integral to the recognition of foreign Ags (23).

IL-12(p70), the heterodimeric cytokine formed from covalently linked chains of IL-12(p40) and IL-12(p35) (24), is responsible for activation of NK cells and differentiation of CD4+ T cells to Th1-like cells (25). The monomer IL-12(p40) is known to act as an antagonist to the IL-12R (26). Although both IL-12(p70) and IL-12(p40) were increased in two-way MLRs as compared with unstimulated control cells, ATL313 only reduced the active heterodimer, IL-12(p70), and did not affect the monomer IL-12(p40). RANTES, a chemokine released primarily by T cells, is known to activate T cells, is responsible for chronic inflammation, and is a powerful chemoattractant for monocytes, NKT and T cells, basophils, eosinophils, and DCs (27, 28, 29). Our results show that ATL313 attenuates secretion of RANTES.

In addition to reducing the release of these cytokines and the expression of CD25, we determined that ATL313 also reduced the cell surface expression of CD40L on CD4+ T cells. CD40L is a costimulatory marker expressed by activated T cells that binds to CD40 on macrophages, working in conjunction with IFN-{gamma} to induce macrophage activation (30, 31). The reduction of both CD40L expression on the cell surface and IFN-{gamma} release from T cells implies that ATL313, in turn, inhibits the activation of monocyte/macrophages. CD40-CD40L is also an important signal pathway for DC and T cell cross-talk, and more important for the initiation of T cells activation, differentiation.

To analyze the mechanisms through which ATL313 mediates CD4+ T cell activation, we stimulated the lymphocytes of a single animal with anti-CD3e mAb to synthetically mimic TCR/CD3 ligation with alloantigens without the dependence on costimulatory markers required by the two-way MLR. In this system, we determined that ATL313 consistently inhibits anti-CD3e mAb-induced proliferation. This suggests that the agonist is able to target the T cell directly and that ATL313 must inhibit a signaling cascade originating with T cells.

Upon binding of TCR/CD3 to its ligand, CD4 and CD45 migrate proximally to the TCR, initiating phosphorylation of the TCR {zeta}-chain ITAMs by receptor-associated Src-family kinases, such as Fyn. The tyrosine kinase Zap70 binds to the phosphorylated {zeta}-chain ITAMs and is then phosphorylated by Lck, another Src family kinase bound to the CD4 molecule. Activated Zap70 in turn phosphorylates the linker for activation of T cells and, through a subsequent signaling cascade, activates MAPK-induced phosphorylation events and a wide variety of transcription factors (see Ref. 32 for a review).

To determine the effects of A2AR agonists on the signaling cascade induced by TCR stimulation, we chose to analyze the phosphorylation of Zap70 in CD4+ T cells. We determined by flow cytometric analysis that anti-CD3e mAb activation over a period of 5 min increases levels of phosphorylated Zap70, and the addition of ATL313 significantly reduces those levels. Because the A2AR agonists are believed to increase levels of intracellular cAMP and consequently the activation of PKA (33), we added a PKA inhibitor, H-89, to the assay along with ATL313 and found that levels of phosphorylated Zap70 were restored. These results provide evidence that A2AR agonists inhibit CD4+ T cell activation by inhibiting the phosphorylation of Zap70 through activation of PKA.

Naive T cells are fully activated after receiving signal 1 through TCR-MHC plus antigenic peptide complex and signal 2 through positive costimulatory molecules (34). CD28:B7 signaling is the best characterized and an important costimulatory pathway for naive T cell activation, including cytokine production, clonal expansion, and prevention of anergy and T cell survival (34, 35). However, blocking CD28:B7 pathway wasn’t effective in inducing tolerance to murine skin and islet transplantation (36, 37). The negative regulatory signal CTLA-4, which is induced after T cell activation, also can bind both B7-1 and B7-2 molecule and play an important role in down-regulating T cell response in organ transplantation (38, 39). New B7 family ligands PD-L1 and PD-L2 are broadly expressed in nonlymphoid tissues, which are important for regulating the effect of memory T cell responses at the site of inflammation. The finding of accelerated allograft rejection by PD-L1 blockade with Abs confirmed that PD-L1 expression is important for inhibiting the rejection process. We used two-way MLR as an in vitro model to investigate the effect of ATL313 effect on these two negative regulatory costimulatory molecules. We found ATL313 induced PD-1 and CTLA-4 expression on CD3/CD4 T cells. Importantly, T cell activation was not necessary for ATL313 to enhance expression of both PD-1 and CTLA-4. These results suggest that ATL313 inhibits T cell activation, proliferation, and cytokine production by activating the cAMP/PKA pathway to reduce IL-2-induced clonal expansion and to induce the negative regulatory molecules CTLA-4 and PD-1.

These results in vitro suggest potent mechanisms by which A2AR agonists can block alloantigen induced immune responses, responses that mediate transplant rejection. Therefore, we tested whether A2AR agonists can block transplant-mediated tissue rejection. We performed full-thickness skin grafts and found that allograft survival was enhanced following ATL313 administration, an effect reversed by ZM241385. These results provide in vivo evidence that A2AR agonists improved graft survival and can delay transplant rejection.

Our previous studies have shown that A2AR-selective agonists attenuate IRI (12), an effect mediated through CD4+ cells (14). Rag-1 KO mice, deficient in lymphocytes, are protected from IRI, whereas the same animals display the effects of IRI when reconstituted with WT CD4+ T lymphocytes, suggesting that these cells contribute to the initiation of the ischemia-reperfusion-induced inflammatory response. The selective adenosine agonist, ATL146e, a compound that is similar to ATL313 but shorter acting, is unable to inhibit IRI when these mice are reconstituted with A2AR KO CD4+ T lymphocytes (14). These data implicate the CD4+ T lymphocyte as an important target through which A2ARs inhibit IRI.

In the current study, we found that A2AR agonists attenuate alloantigen recognition through effects mediated by A2ARs expressed not only on T cells but on APCs as well. Furthermore, A2AR agonists have direct inhibitory effects on CD4+ T cell activation. Thus, these in vitro and in vivo results indicate that A2AR agonists attenuate alloantigen recognition and could induce peripheral T cell tolerance, resulting in improved graft survival. Furthermore, to our knowledge, this is the first demonstration that A2AR agonists delay transplant rejection. The marked efficacy of A2AR agonists in protecting kidneys from IRI in prior studies and allograft rejection in the current studies makes these compounds ideal candidates for induction therapy following organ transplantation.


   Acknowledgments
 
We acknowledge Drs. Timothy Mcdonald (Department of Chemistry, University of Virginia) and Jayson Rieger (Adenosine Therapeutics, Charlottesville, VA) for the gift of ATL313 and Dr. Jiang-Fan Chen (Department of Neurology, Boston University School of Medicine, Boston, MA) for A2AKO mice. We are grateful to Hong Ye and Kailo Schlegel for discussions and technical assistance and Dr. Diane L. Rosin (Department of Pharmacology, University of Virginia) for careful reading of the manuscript and helpful discussions.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
M. D. Okusa and J. Linden own equity in Adenosine Therapeutics, which provided ATL313 for this study.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants DK56223, DK62324, DK58413, and HL37942. Back

2 C.P.S. and L.L. participated equally in this study. Back

3 Address correspondence and reprint requests to Dr. Mark D. Okusa, Division of Nephrology, Box 800133, University of Virginia Health System, Charlottesville, VA 22908. E-mail address: mdo7y{at}virginia.edu Back

4 Abbreviations used in this paper: IRI, ischemia-reperfusion injury; A2AR, adenosine 2A receptor; DC, dendritic cell; KO, knockout; MCF, mean channel fluorescence; PD, programmed death; PKA, protein kinase A; WT, wild type. Back

Received for publication January 30, 2006. Accepted for publication January 10, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Sayegh, M. H., L. A. Turka. 1998. The role of T cell costimulatory activation pathways in transplant rejection. N. Engl. J. Med. 338: 1813-1821. [Free Full Text]
  2. Rothstein, D. M., M. H. Sayegh. 2003. T cell costimulatory pathways in allograft rejection and tolerance. Immunol. Rev. 196: 85-108. [Medline]
  3. Greenwald, R. J., Y. E. Latchman, A. H. Sharpe. 2002. Negative co-receptors on lymphocytes. Curr. Opin. Immunol. 14: 391-396. [Medline]
  4. Sho, M., A. D. Salama, A. Yamada, N. Najafian, M. H. Sayegh. 2001. Physiologic regulation of alloimmune responses in vivo: the role of CTLA4 and Th1/Th2 cytokines. Transplant. Proc. 33: 3826-3828. [Medline]
  5. Freeman, G. J., A. J. Long, Y. Iwai, K. Bourque, T. Chernova, H. Nishimura, L. J. Fitz, N. Malenkovich, T. Okazaki, M. C. Byrne, et al 2000. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J. Exp. Med. 192: 1027-1034. [Abstract/Free Full Text]
  6. Olah, M. E., G. L. Stiles. 1995. Adenosine receptor subtypes: characterization and therapeutic regulation. Ann. Rev. Pharmacol. Toxicol. 35: 581-606. [Medline]
  7. Linden, J.. 2001. Molecular approach to adenosine receptors: receptor mediated mechanisms of tissue protection. Annu. Rev. Pharmacol. Toxicol. 41: 775-787. [Medline]
  8. Perico, N., D. Cattaneo, M. H. Sayegh, G. Remuzzi. 2004. Delayed graft function in kidney transplantation. Lancet 364: 1814-1827. [Medline]
  9. Reece, T. B., J. D. Davis, D. O. Okonkwo, T. S. Maxey, P. I. Ellman, X. Li, J. Linden, C. G. Tribble, I. L. Kron, J. A. Kern. 2004. Adenosine A2A analogue reduces long-term neurologic injury after blunt spinal trauma. J. Surg. Res. 121: 130-134. [Medline]
  10. Day, Y. J., L. Huang, H. Ye, J. Linden, M. D. Okusa. 2005. Renal ischemia-reperfusion injury and adenosine 2A receptor-mediated tissue protection: role of macrophages. Am. J. Physiol. 288: F722-F731.
  11. Glover, D. K., L. M. Riou, M. Ruiz, G. W. Sullivan, J. Linden, J. M. Rieger, T. L. Macdonald, D. D. Watson, G. A. Beller. 2005. Reduction of infarct size and post-ischemic inflammation from a highly selective adenosine A2A receptor agonist, ATL-146e, in reperfused canine myocardium. Am. J. Physiol. 288: H1851-H1858.
  12. Okusa, M. D., J. Linden, T. Macdonald, L. Huang. 1999. Selective A2A-adenosine receptor activation during reperfusion reduces ischemia-reperfusion injury in rat kidney. Am. J. Physiol. 277: F404-F412. [Medline]
  13. Day, Y.-J., L. Huang, M. J. McDuffie, D. L. Rosin, H. Ye, J. F. Chen, M. A. Schwarzschild, J. S. Fink, J. Linden, M. D. Okusa. 2003. Renal protection from ischemia mediated by A2A adenosine receptors on bone marrow-derived cells. J. Clin. Invest. 112: 883-891. [Medline]
  14. Day, Y.-J., L. Huang, H. Ye, L. Li, J. Linden, M. D. Okusa. 2006. Renal ischemia-reperfusion and adenosine 2A receptor-mediated tissue protection: the role of CD4+ T cells and interferon {gamma}. J. Immunol. 176: 3108-3114. [Abstract/Free Full Text]
  15. Lappas, C. M., J. M. Rieger, J. Linden. 2005. A2A adenosine receptor induction inhibits IFN-{gamma} production in murine CD4+ T cells. J. Immunol. 174: 1073-1080. [Abstract/Free Full Text]
  16. Chen, J. F., Z. Huang, J. Ma, J. Zhu, R. Moratalla, D. Standaert, M. A. Moskowitz, J. S. Fink, M. A. Schwarzschild. 1999. Nov 1. A2A adenosine receptor deficiency attenuates brain injury induced by transient focal ischemia in mice. J. Neurosci. 19: 9192-9200. [Abstract/Free Full Text]
  17. Copeland, N. G., D. J. Gilbert, N. A. Jenkins, J. H. Nadeau, J. T. Eppig, L. J. Maltais, J. C. Miller, W. F. Dietrich, R. G. Steen, S. E. Lincoln, et al 1993. Genome maps IV 1993 wall chart. Science 262: 67-82. [Free Full Text]
  18. Dietrich, W. F., J. C. Miller, R. G. Steen, M. Merchant, D. Damron, R. Nahf, A. Gross, D. C. Joyce, M. Wessel, R. D. Dredge, et al 1994. A genetic map of the mouse with 4,006 simple sequence length polymorphisms. Nat. Genet. 7: 220-245. [Medline]
  19. Dietrich, W. F., J. Miller, R. Steen, M. A. Merchant, D. Damron-Boles, Z. Husain, R. Dredge, M. J. Daly, K. A. Ingalls, T. J. O’Connor. 1996. A comprehensive genetic map of the mouse genome. Nature 380: 149-152. [Medline]
  20. Potian, J. A., H. Aviv, N. M. Ponzio, J. S. Harrison, P. Rameshwar. 2003. Veto-like activity of mesenchymal stem cells: functional discrimination between cellular responses to alloantigens and recall antigens. J. Immunol. 171: 3426-3434. [Abstract/Free Full Text]
  21. Cantrell, D. A., K. A. Smith. 1984. The interleukin-2 T cell system: a new cell growth model. Science 224: 1312-1316. [Abstract/Free Full Text]
  22. Zicari, A., M. Lipari, L. Di Renzo, A. Longo, G. Antonelli, G. M. Pontieri. 1992. In vivo and in vitro macrophage activation induced by IFN-{gamma} spontaneously released by spleen cells from tumor bearing mice. J. Biol. Regul. Homeostatic Agents 6: 65-72.
  23. Vegh, Z., P. Wang, F. Vanky, E. Klein. 1993. Increased expression of MHC class I molecules on human cells after short time IFN-{gamma} treatment. Mol. Immunol. 30: 849-854. [Medline]
  24. Gubler, U., A. O. Chua, D. S. Schoenhaut, C. M. Dwyer, W. McComas, R. Motyka, N. Nabavi, A. G. Wolitzky, P. M. Quinn, P. C. Familletti, et al 1991. Coexpression of two distinct genes is required to generate secreted bioactive cytotoxic lymphocyte maturation factor. Proc. Natl. Acad. Sci. USA 88: 4143-4147. [Abstract/Free Full Text]
  25. Trinchieri, G.. 1994. Interleukin-12: a cytokine produced by antigen-presenting cells with immunoregulatory functions in the generation of T-helper cells type 1 and cytotoxic lymphocytes. Blood 84: 4008-4027. [Free Full Text]
  26. Germann, T., E. Rude, F. Mattner, M. K. Gately. 1995. The IL-12 p40 homodimer as a specific antagonist of the IL-12 heterodimer. Immunol. Today 16: 500-501. [Medline]
  27. Meurer, R., G. Van Riper, W. Feeney, P. Cunningham, D. Hora, Jr, M. S. Springer, D. E. MacIntyre, H. Rosen. 1993. Formation of eosinophilic and monocytic intradermal inflammatory sites in the dog by injection of human RANTES but not human monocyte chemoattractant protein 1, human macrophage inflammatory protein 1{alpha}, or human interleukin 8. J. Exp. Med. 178: 1913-1921. [Abstract/Free Full Text]
  28. Schall, T. J.. 1991. Biology of the RANTES/SIS cytokine family. Cytokine 3: 165-183. [Medline]
  29. Schall, T. J., K. Bacon, K. J. Toy, D. V. Goeddel. 1990. Selective attraction of monocytes and T lymphocytes of the memory phenotype by cytokine RANTES. Nature 347: 669-671. [Medline]
  30. Stout, R. D., J. Suttles, J. Xu, I. S. Grewal, R. A. Flavell. 1996. Impaired T cell-mediated macrophage activation in CD40 ligand-deficient mice. J. Immunol. 156: 8-11. [Abstract]
  31. Grewal, I. S., R. A. Flavell. 1996. A central role of CD40 ligand in the regulation of CD4+ T cell responses. Immunol. Today 17: 410-414. [Medline]
  32. Qian, D., A. Weiss. 1997. T cell antigen receptor signal transduction. Curr. Opin. Cell Biol. 9: 205-212. [Medline]
  33. Skalhegg, B. S., K. Tasken. 2000. Specificity in the cAMP/PKA signaling pathway: differential expression, regulation, and subcellular localization of subunits of PKA. Front. Biosci. 5: D678-D693. [Medline]
  34. Linsley, P. S., J. A. Ledbetter. 1993. The role of the CD28 receptor during T cell responses to antigen. Annu. Rev. Immunol. 11: 191-212. [Medline]
  35. Dahl, A. M., C. Klein, P. G. Andres, C. A. London, M. P. Lodge, R. C. Mulligan, A. K. Abbas. 2000. Expression of bcl-XL restores cell survival, but not proliferation off effector differentiation, in CD28-deficient T lymphocytes. J. Exp. Med. 191: 2031-2038. [Abstract/Free Full Text]
  36. Larsen, C. P., E. T. Elwood, D. Z. Alexander, S. C. Ritchie, R. Hendrix, C. Tucker-Burden, H. R. Cho, A. Aruffo, D. Hollenbaugh, P. S. Linsley, et al 1996. Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways. Nature 381: 434-438. [Medline]
  37. Trambley, J., A. W. Bingaman, A. Lin, E. T. Elwood, S. Y. Waitze, J. Ha, M. M. Durham, M. Corbascio, S. R. Cowan, T. C. Pearson, C. P. Larsen. 1999. Asialo GM1+CD8+ T cells play a critical role in costimulation blockade-resistant allograft rejection. J. Clin. Invest. 104: 1715-1722. [Medline]
  38. Ansari, M. J., A. D. Salama, T. Chitnis, R. N. Smith, H. Yagita, H. Akiba, T. Yamazaki, M. Azuma, H. Iwai, S. J. Khoury, et al 2003. The programmed death-1 (PD-1) pathway regulates autoimmune diabetes in nonobese diabetic (NOD) mice. J. Exp. Med. 198: 63-69. [Abstract/Free Full Text]
  39. Salama, A. D., T. Chitnis, J. Imitola, M. J. Ansari, H. Akiba, F. Tushima, M. Azuma, H. Yagita, M. H. Sayegh, S. J. Khoury. 2003. Critical role of the programmed death-1 (PD-1) pathway in regulation of experimental autoimmune encephalomyelitis. J. Exp. Med. 198: 71-78. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
S. P. Hilchey, J. J. Kobie, M. R. Cochran, S. Secor-Socha, J.-C. E. Wang, O. Hyrien, W. R. Burack, T. R. Mosmann, S. A. Quataert, and S. H. Bernstein
Human Follicular Lymphoma CD39+-Infiltrating T Cells Contribute to Adenosine-Mediated T Cell Hyporesponsiveness
J. Immunol., November 15, 2009; 183(10): 6157 - 6166.
[Abstract] [Full Text] [PDF]

Home page
 Ann. Thorac. Surg.Home page
C. L. Lau, Y. Zhao, I. L. Kron, M. H. Stoler, V. E. Laubach, G. Ailawadi, and J. Linden
The Role of Adenosine A(2A) Receptor Signaling in Bronchiolitis Obliterans.
Ann. Thorac. Surg., October 1, 2009; 88(4): 1071 - 1078.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
P. E. Zarek, C.-T. Huang, E. R. Lutz, J. Kowalski, M. R. Horton, J. Linden, C. G. Drake, and J. D. Powell
A2A receptor signaling promotes peripheral tolerance by inducing T-cell anergy and the generation of adaptive regulatory T cells
Blood, January 1, 2008; 111(1): 251 - 259.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sevigny, C. P.
Right arrow Articles by Okusa, M. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sevigny, C. P.
Right arrow Articles by Okusa, M. D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS